The ruminant diet normally contains little or no vitamin B12. Rumen microorganisms are known to synthesize vitamin B12 as well as a number of analogues of the vitamin. Research being conducted by the principal investigator has resulted in quantitative estimates of B12 production in the rumen and absorption from the small intestine of the sheep. Several dietary factors have now been shown to influence production, and presumptive evidence of the presence of B12 analogues in serum of the ruminant on certain diets has been produced. The objectives of the proposed research are to study the effect of riboflavin on vitamin B12 production by rumen microorganisms, to identify the B12 analogues believed present in sheep and cow serum, and to test the hypothesis that there is an association between the incidence of bovine ketosis and the presence or level of B12 analogues in serum. Surgically-modified sheep with re-entrant intestinal cannulae permitting collection and sampling of digesta over 24-hour periods will be used to study production and absorption. A non-specific measure of analogues will be obtained by using two B12 assays - one of which responds almost only to vitamin B12, the other of which responds to both B12 and analogues. Identification of analogues will be accomplished by electrophoretic separation and subsequent bioautography. Extracts of sera from ketotic and control cows will be subjected to these techniques to test the hypothesis in question.